ABSTRACTHypoxia induced intestinal barrier injury, microbial translocation, and local/systemic inflammation may contribute to high‐altitude associated gastrointestinal complications or symptoms of acute mountain sickness (AMS). Therefore, we tested the hypothesis that six‐hours of hypobaric hypoxia increases circulating markers of intestinal barrier injury and inflammation. A secondary aim was to determine if the changes in these markers were different between those with and without AMS. Thirteen participants were exposed to six hours of hypobaric hypoxia, simulating an altitude of 4572 m. Participants completed two 30‐minute bouts of exercise during the early hours of hypoxic exposure to mimic typical activity required by those at high altitude. Pre‐ and post‐exposure blood samples were assessed for circulating markers of intestinal barrier injury and inflammation. Data below are presented as mean ± standard deviation or median [interquartile range]. Intestinal fatty acid binding protein (Δ251 [103–410] pg•mL⁻¹; p = 0.002, d = 0.32), lipopolysaccharide binding protein (Δ2 ± 2.4 μg•mL⁻¹; p = 0.011; d = 0.48), tumor necrosis factor‐α (Δ10.2 [3–42.2] pg•mL⁻¹; p = 0.005; d = 0.25), interleukin‐1β (Δ1.5 [0–6.7] pg•mL⁻¹ p = 0.042; d = 0.18), and interleukin‐1 receptor agonist (Δ3.4 [0.4–5.2] pg•mL⁻¹p = 0.002; d = 0.23) increased from pre‐ to post‐hypoxia. Six of the 13 participants developed AMS; however, the pre‐ to post‐hypoxia changes for each marker were not different between those with and without AMS (p > 0.05 for all indices). These data provide evidence that high altitude exposures can lead to intestinal barrier injury, which may be an important consideration for mountaineers, military personnel, wildland firefighters, and athletes who travel to high altitudes to perform physical work or exercise.